Recently, I perform a number of pflB-focA gene knock-out using lamda RED recombination in Escherichia coli strain W1485. But I have a hard problem.Below is my experiment:1. PCR amplify linear fragment from both pKD3 and pKD4Purify PCR product by gel.2. Make target W1485strain maintaining pKD46 by growing at 30°C with 10 mM L-arabinose (E. coli was induced for just 1 h before harvesting)3. Transform E. coli using Calcium Chlorid.4. Plate the transformation culture on LB plates supplemented with chloramphenicol (8ug/ml) or kanamycin(40ug/ml).

Here is the results:1. I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol. 2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain. Can anyone tell me what could be the problem.During RED recombination, is electroporation necessary?